Bacterial antibiotic efflux transporters are important players in conferring intrinsic and acquired resistance to antimicrobials. Campylobacterjejuni, a leading bacterial cause of foodborne diarrhea in the United States and an agent included in the NIAID Category B Priority Pathogens list, harbors multiple antibiotic efflux transporters of different families. During the previous grant period, we determined the function and regulation of two efflux pumps (CmeABC and CmeDEF) of the resistance-nodulation-division (RND) family in Campylobacter. Our findings indicate that the efflux system not only contributes to antimicrobial resistance, but also has important physiological functions in facilitating Campylobacter colonization in the intestinal tract of animals. We have also found that CmeR, a transcriptional factor, represses cmeABC and that bile salts (normally present in the gut) induce the expression of cmeABC by inhibiting the binding of CmeR to the promoter of cmeABC. Our recent preliminary studies also strongly suggest that CmeR is a pleiotropic regulator and modulates the expression of the MF (major facilitator) and MATE (multidrug and toxic compound extrusion) transporters as well as the C4-dicarboxylate transporters potentially involved in Campylobacter adaptation to the oxygen-limited environment in animal intestine. These findings indicate that the antibiotic efflux system is co-regulated by CmeR with other physiological processes and plays important roles in antimicrobial resistance and in facilitating Campylobacter adaptation to environmental changes. Despite these recent advances, the majority of the CmeR-regulated efflux transporters in Campylobacter have not been functionally characterized, and the molecular mechanisms governing the expression of the transporters and the structural basis of CmeR regulation remain to be determined. To close these important gaps in our understanding of the active efflux system in Campylobacter, we plan to pursue 3 specific aims in this renewal application to 1) determine the regulatory mechanisms and functions of the MF and MATE transporters in C. jejuni, 2) define the regulation and role of the C4-dicarboxylate transport system in facilitating Campylobacter adaptation to oxygen-limited conditions, and 3) elucidate the structural basis of CmeR regulation and the mechanisms of bile induction using X-ray crystallography. The proposed studies take advantage of unique resources available in our laboratories and utilize contemporary molecular, genetic, and biochemical approaches as well as an established animal model. Once completed, the proposed work together with the studies conducted in the previous grant period will reveal novel information on the functions and regulatory mechanisms of antibiotic efflux transporters in bacteria. The findings will help to identify potential molecular targets for the control and treatment of antibiotic resistant Campylobacter.